Probe, probe set, probe carrier, and testing method

ABSTRACT

A probe, a set of probes, and a probe carrier on which the probe or the set of probes is immobilized, are provided for classification of fungus species. The probe or the set of probes is capable of collectively detecting fungus of the same species and distinguishingly detecting those fungus from fungus of other species. The probe is an oligonucleotide probe for detecting a pathogenic fungus DNA and includes at least one of base sequences of SEQ ID NOS. 1 to 4 and mutated sequences thereof.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a probe and a probe set for detecting aDNA of a pathogenic fungus, Candida kefyr, which are useful fordetection and identification of a causative fungus of an infectiousdisease, and to a probe carrier on which the probe or the probe set isimmobilized. The present invention also relates to a DNA testing methodand a DNA testing kit using the same.

2. Description of the Related Art

Heretofore, reagents for and methods of quickly and accurately detectingpathogenic fungi in a sample have been proposed. For instance, JapanesePatent Application Laid-Open No. H08-089254 discloses oligonucleotideshaving specific base sequences, which can be used as probes and primersfor detecting pathogenic fungi of candidiasis and aspergillosis, and amethod of detecting target fungi using such oligonucleotides. Inaddition, the same patent document also discloses a primer set used forPCR amplifying a plurality of target fungi in common. Further, the samepatent document also discloses a method of identifying fungus species ina sample comprising subjecting a plurality of target fungi in the sampleto PCR amplification using the primer set, and then detecting thesequence portions specific to the respective fungi by a hybridizationassay using the probes specific to the respective fungi.

On the other hand, a method capable of simultaneously detecting aplurality of oligonucleotides having different base sequences is known.The method uses a probe array in which probes having sequencescomplementary to the respective base sequences are arranged at intervalson a solid phase (Japanese Patent Application Laid-Open No.2004-313181).

SUMMARY OF THE INVENTION

However, it is not easy to establish a probe which specifically detectsa DNA of a pathogenic fungus in a sample. The sample may contain notonly the DNA of the pathogenic fungus but also DNAs of other pathogenicfungi. Thus, it is not easy to establish a probe that specificallydetects a DNA of a pathogenic fungus which is less susceptible to theinfluence of the presence of DNAs of other pathogenic fungi (i.e. crosscontamination). Under such circumstances, the inventors of the presentinvention have conducted investigation on probes for detecting thefollowing pathogenic fungi. The object of the investigation was toobtain probes capable of detecting a DNA of a target pathogenic funguswith a high degree of accuracy even for an analyte containing DNAs of aplurality of fungi with a less cross-contamination level. As a result, aplurality of probes capable of detecting a pathogenic fungus DNA with ahigh degree of accuracy were finally obtained.

Candida Kefyr

A first object of the present invention is to provide a probe and aprobe set which are capable of accurately identifying the DNA of atarget fungus.

Another object of the present invention is to provide a probe carrierwhich is capable of accurately identifying a target fungus from a samplein which various kinds of fungi may exist together.

Still another object of the present invention is to provide a DNAtesting method for a pathogenic fungus, which can more quickly and moreaccurately detect a target fungus from a sample when various kinds offungi exist in the sample, and to provide a kit for the testing method.

The probe of the present invention for detecting a DNA of Candida kefyrwhich is a pathogenic fungus includes one of the following basesequences (1) to (5):

(1) gcggccagttcttgattctctgc (SEQ ID NO. 1) or a complementary sequencethereof;

(2) agctcgtctctccagtggacataaac (SEQ ID NO. 2) or a complementarysequence thereof;

(3) ttgaaagtggctagccgttgcc (SEQ ID NO. 3) or a complementary sequencethereof;

(4) tcgtggtaagcttgggtcatagagac (SEQ ID NO. 4) or a complementarysequence thereof; and

(5) a mutated sequence which is obtained by deletion, substitution, oraddition of a base on one of the sequences of SEQ ID NOS. 1 to 4 and thecomplementary sequences thereof in a range that the mutated sequenceretains a function as the probe.

In addition, the probe set of the present invention for detecting a DNAof Candida kefyr which is a pathogenic fungus includes at least two ofthe following probes (A) to (P):

(A) a probe including a base sequence represented bygcggccagttcttgattctctgc (SEQ ID NO. 1);

(B) a probe including a base sequence represented byagctcgtctctccagtggacataaac (SEQ ID NO. 2);

(C) a probe including a base sequence represented byttgaaagtggctagccgttgcc (SEQ ID NO. 3);

(D) a probe including a base sequence represented bytcgtggtaagcttgggtcatagagac (SEQ ID NO. 4);

(E) a probe including a complementary sequence of SEQ ID NO. 1;

(F) a probe including a complementary sequence of SEQ ID NO. 2;

(G) a probe including a complementary sequence of SEQ ID NO. 3;

(H) a probe including a complementary sequence of SEQ ID NO. 4;

(I) a probe including a mutated sequence obtained by deletion,substitution or addition of a base on SEQ ID NO. 1 within a range thatthe mutated sequence retains a function as the probe;

(J) a probe including a mutated sequence obtained by deletion,substitution or addition of a base on SEQ ID NO. 2 within a range thatthe mutated sequence retains the function as the probe;

(K) a probe including a mutated sequence obtained by deletion,substitution or addition of a base on SEQ ID NO. 3 within a range thatthe mutated sequence retains the function as the probe;

(L) a probe including a mutated sequence obtained by deletion,substitution or addition of a base on SEQ ID NO. 4 within a range thatthe mutated sequence retains the function as the probe;

(M) a probe including a mutated sequence obtained by deletion,substitution or addition of a base on the complementary sequence of SEQID NO. 1 within a range that the mutated sequence retains the functionas the probe;

(N) a probe including a mutated sequence obtained by deletion,substitution or addition of a base on the complementary sequence of SEQID NO. 2 within a range that the mutated sequence retains the functionas the probe;

(O) a probe including a mutated sequence obtained by deletion,substitution or addition of a base on the complementary sequence of SEQID NO. 3 within a range that the mutated sequence retains the functionas the probe; and

(P) a probe including a mutated sequence obtained by deletion,substitution or addition of a base on the complementary sequence of SEQID NO. 4 within a range that the mutated sequence retains the functionas the probe.

The probe carrier of the present invention includes a carrier on whichthe probe or each of the plurality of probes constituting the probe setmentioned above is placed on a carrier.

In the probe carrier of the present invention, at least one of theprobes (A) to (P) is immobilized on a carrier, and when a plurality ofprobes is to be immobilized, the respective probes are placed whilebeing isolated from each other.

The method of detecting DNA of Candida kefyr in a sample by using aprobe carrier according to the present invention includes:

(i) reacting the sample with the probe carrier; and

(ii) detecting a reaction intensity of a probe on the probe carrierreacted with a nucleic acid contained in the sample.

The kit for detecting a pathogenic fungus of the present invention is akit for detecting a DNA of Candida kefyr, including at least one of theprobes (A) to (P) or a probe carrier on which at least one of thoseprobes is immobilized and a reagent for detecting a reaction of a probewith a target nucleic acid.

According to the present invention, when a specimen is infected with thecausative fungus mentioned above, the fungus can be more quickly andprecisely identified from the specimen even if the specimen issimultaneously and complexly infected with other fungi in addition tothe above-mentioned fungus. In particular, Candida kefyr can be detectedwhile precisely distinguishing it from any of fungi of other Candidaspecies, fungi of Trichosporon species, fungi of Cryptococcus species,fungi of Aspergillus species, fungi of Epidermophyton species, fungi ofArthroderma species, and fungi of Trichophyton species, which may bemore likely to cause cross contamination.

Further features of the present invention will become apparent from thefollowing description of exemplary embodiments with reference to theattached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram illustrating a 1st PCR protocol.

FIG. 2 is a diagram illustrating a 2nd PCR protocol.

FIG. 3 is a diagram illustrating a protocol of hybridization.

DESCRIPTION OF THE EMBODIMENTS

The present invention provides an oligonucleotide probe for identifyinga pathogenic fungus (hereinafter, simply referred to as a probe) and aprobe set including a combination of two or more probes. The use of sucha probe or a probe set allows the detection of the following funguswhich will cause inflammation by infection.

(Fungus Name)

Candida Kefyr

The probe of the present invention can react with the DNA sequence of aninternal transcribed spacer (ITS) of the pathogenic fungus or a nucleicacid having a base sequence specific to the ITS region contained in asample. The probe can be selected from the oligonucleotides having thefollowing base sequences:

(A) a probe including a base sequence represented bygcggccagttcttgattctctgc (SEQ ID NO. 1);

(B) a probe including a base sequence represented byagctcgtctctccagtggacataaac (SEQ ID NO. 2);

(C) a probe including a base sequence represented byttgaaagtggctagccgttgcc (SEQ ID NO. 3);

(D) a probe including a base sequence represented bytcgtggtaagcttgggtcatagagac (SEQ ID NO. 4)

(E) a probe including a complementary sequence of the base sequencerepresented by SEQ ID NO. 1;

(F) a probe including a complementary sequence of the base sequencerepresented by SEQ ID NO. 2;

(G) a probe including a complementary sequence of the base sequencerepresented by SEQ ID NO. 3;

(H) a probe including a complementary sequence of the base sequencerepresented by SEQ ID NO. 4;

(I) a probe including a mutated sequence obtained by deletion,substitution or addition of a base on SEQ ID NO. 1 in a range that themutated sequence retains a function as the probe;

(J) a probe including a mutated sequence obtained by deletion,substitution or addition of a base on SEQ ID NO. 2 in a range that themutated sequence retains the function as the probe;

(K) a probe including a mutated sequence obtained by deletion,substitution or addition of a base on SEQ ID NO. 3 in a range that themutated sequence retains the function as the probe;

(L) a probe including a mutated sequence obtained by deletion,substitution or addition of a base on SEQ ID NO. 4 in a range that themutated sequence retains the function as the probe;

(M) a probe including a mutated sequence obtained by deletion,substitution or addition of a base on SEQ ID NO. 1 in a range that themutated sequence retains the function as the probe;

(N) a probe including a mutated sequence obtained by deletion,substitution or addition of a base on the complementary sequence of SEQID NO. 2 in a range that the mutated sequence retains the function asthe probe;

(O) a probe including a mutated sequence obtained by deletion,substitution or addition of a base on the complementary sequence of SEQID NO. 3 in a range that the mutated sequence retains the function asthe probe; and

(P) a probe including a mutated sequence obtained by deletion,substitution or addition of a base on the complementary sequence of SEQID NO. 4 in a range that the mutated sequence retains the function asthe probe.

The probe set can be prepared using two or more of those probes. The ITSregion of DNA of the pathogenic fungus can be sufficiently detectedusing the probe set of the present invention.

The mutated sequences include any mutation as far as it does not impairthe function of the probe, or any mutation as far as it hybridizes witha nucleic acid sequence of interest as a detection target. Of those, itis desirable to include any mutation as far as it can hybridize with anucleic acid sequence of interest as a detection target under stringentconditions. Preferable hybridization conditions confining the mutationinclude those represented in examples as described below. Here, the term“detection target” used herein may be one included in a sample to beused in hybridization, which may be a unique base sequence to thepathogenic fungus, or may be a complementary sequence to the unique basesequence. Further, the variation may be a mutated sequence obtained bydeletion, substitution, or addition of at least one base as far as itretains function as the probe.

The characteristics of those probes depend on the specificities of therespective probe sequences to the target nucleic acid sequence as a testobject. The specificity of the probe sequence can be evaluated from thedegree of coincidence between the base sequence thereof and the targetnucleic acid sequence and the melting temperature of a duplex of thetarget nucleic acid sequence and the probe sequence. In addition, whenthe probe is constituting a probe set, the performance of the probe alsodepends on a difference between the melting temperature of the probe andthe melting temperature of another probe sequence in the probe set.

For designing these probe sequences, base sequences with highspecificities to the same fungus species but with less variation betweendifferent strains in the same species may be selected. In addition, aregion can be selected to have three or more unmatched bases withrespect to the sequence of the fungus species other than the fungusspecies of interest. Further, it is designed such that the differencebetween the melting temperature of the duplex of the probe sequence andthe sequence of the fungus species of interest and the meltingtemperature of the duplex of the probe sequence and the sequence of anyfungus species other than the species of interest is 10° C. or more.Further, it is designed such that any base is deleted from or added tothe highly specific region to hold the melting temperatures of therespective probes immobilized on the same carrier within a predeterminedtemperature range.

The experiment conducted by the inventors of the present invention hasrevealed that the attenuation of hybridization intensity may be smallwhen 80% or more of the continuous base sequence is conserved.Therefore, of the probe sequence disclosed in the present application,any mutated sequence may retain the functions of the probe as long asthe mutated sequence conserves 80% or more of the continuous basesequence.

Those probe sequences are specific to the DNA sequence of the ITS regionof the fungus of interest, so a sufficient hybridization sensitivity tothe sequence is expected. In addition, those probe sequences aredesigned so that it will bring a good result by forming a stable hybridin a hybridization reaction with a target specimen even in a state ofbeing immobilized on a carrier. Further, those probes are designed sothat the melting temperature thereof falls within a predeterminedtemperature range.

Further, those probe sequences are designed so that fungus species canbe determined with a combination of specific portions withoutdetermining the sequence of the entire ITS region.

Further, a probe carrier (e.g., DNA chip), on which the probe fordetecting a pathogenic fungus of the present invention is immobilized,can be obtained by supplying the probe on a predetermined position onthe carrier and immobilizing the probe thereon. Various methods can beused for the supply of probe to the carrier. For example, a method whichcan be suitably used is to keep the probe in a state of beingimmobilized on the carrier through a chemical bonding (e.g., covalentbonding) and a liquid containing the probe is then provided on apredetermined position by an inkjet method. Such a method allows theprobe to be hardly detached from the carrier and exerts an additionaleffect of improving the sensitivity. In other words, when a stampingmethod conventionally called a Stanford method in common use is employedto make a DNA chip, the resultant DNA chip has a disadvantage in thatthe applied DNA tends to be peeled off. In addition, one of the methodsof producing DNA chips is to carry out the placement of probes by thesynthesis of DNA on the surface of a carrier (e.g., Oligonucleotidearray manufactured by Affymetrix Co., Ltd.). In the method ofsynthesizing the probe on the carrier, it is difficult to controlsynthesis amount for each probe. Thus, the amount of immobilized probeper immobilization area (spot) for each probe tends to vary considerablyfrom one another. Such variations in amounts of the respectiveimmobilized probes may cause inaccurate evaluation on the results of thedetection with those probes. Based on this fact, the probe carrier ofthe present invention is preferably prepared using the inkjet method.The inkjet method as described above has an advantage in that the probecan be stably immobilized on the carrier and hardly detaching from thecarrier to efficiently provide a probe carrier which can be detectedwith high sensitivity and high accuracy.

For using a plurality of probes immobilized on a carrier, the probes aredesigned to have a given melting temperature to simplify the protocol ofhybridization.

Hereinafter, preferred embodiments of the present invention will bedescribed in further detail with reference to the attached drawings.

First, the terms used herein will be elucidated as follows:

The term “specimen” represents one obtained as a target of anexamination. The term “analyte” represents one prepared from the analyteto contain DNA or nucleic acid fragments. The term “sample” represents atarget to be reacted with a probe. The “sample” includes the specimenwhen the specimen is directly reacted with a probe. The “sample”includes the analyte when the analyte prepared from the specimen isdirectly reacted with a probe.

The specimen to be tested using probe carriers (e.g., DNA chips) inwhich the probes of the present invention are immobilized on carriersinclude those originated from humans and animals such as domesticanimals. For example, the test object is any of those which may containfungi including: any body fluids such as blood, cerebrospinal fluid,expectorated sputum, gastric juice, vaginal discharge, and oral mucosalfluid; tissues such as skin, fingernails, and hair; and excretions suchas urine and feces. All media, which can be contaminated with fungi, canbe also subjected to a test using the DNA chip. The media include: food,drink water, and water in the natural environment such as hot springwater, which may cause food poisoning by contamination; filters of aircleaners and the like; and the like. Animals and plants, which should bequarantined in import/export, are also used as specimens of interest.

When the specimen as described above can be directly used in reactionwith the DNA chip, it is used as a sample to react with the DNA chip andthe result of the reaction is then analyzed. Further, when the specimencannot be directly reacted with the DNA chip, the analyte was subjectedto extraction, purification, and other procedures for obtaining a targetsubstance as required and then provided as a sample to carry out areaction with the DNA chip. For instance, when the specimen contains atarget nucleic acid, an extract, which may be assumed to contain such atarget nucleic acid, is prepared from a specimen, and then washed,diluted, or the like to obtain an analyte as a sample followed byreaction with the DNA chip. Further, when a target nucleic acid isincluded in a specimen obtained by carrying out various amplificationprocedures such as PCR amplification, the target nucleic acid may be asample for being amplified and then reacted with a DNA chip. Suchanalytes of amplified nucleic acids include the following.

(a) An amplified analyte prepared by using a PCR-reaction primerdesigned for detecting ITS region.

(b) An amplified analyte prepared by an additional PCR reaction or thelike from a PCR-amplified product.

(c) An analyte prepared by an amplification method other than PCR.

(d) An analyte labeled for visualization by any of various labelingmethods.

Further, a carrier used for preparing a probe carrier such as a DNA chipmay be any of those that satisfy the property of carrying out a solidphase-liquid phase reaction of interest. Examples of the carrierinclude: flat substrates such as a glass substrate, a plastic substrate,and a silicon wafer; a three-dimensional structure having an irregularsurface; a spherical body such as a bead; and rod-, cord-, andthread-shaped structures. The surface of the carrier may be processedsuch that a probe can be immobilized thereon. In particular, a carrierprepared by introducing a functional group to its surface to makechemical reaction possible has a preferable form from the viewpoint ofreproducibility because the probe is stably bonded in the process ofhybridization reaction.

Various methods can be employed for the immobilization of probes. Anexample of the method of using a combination of a maleimide group and athiol (—SH) group. In this method, a thiol (—SH) group is bonded to theterminal of a probe, and a process is executed in advance to make thecarrier (solid phase) surface have a maleimide group. Accordingly, thethiol group of the probe supplied to the carrier surface reacts with themaleimide group on the carrier surface to form a covalent bond, wherebythe probe is immobilized.

First, introduction of the maleimide group can utilize a process ofallowing a reaction of a glass substrate with an aminosilane couplingagent and then introducing an maleimide group onto the glass substrateby a reaction of the amino group with an EMCS reagent(N-(6-maleimidocaproyloxy)succinimide, manufactured by DojindoLaboratories). Introduction of the thiol group to a DNA can be carriedout using 5′-Thiol-Modifier C6 (manufactured by Glen ResearchCorporation) when the DNA is synthesized by an automatic DNAsynthesizer. Instead of the combination of a thiol group and a maleimidegroup, a combination of, e.g., an epoxy group (on the solid phase) andan amino group (nucleic acid probe terminal), can also be used as acombination of functional groups to be used for immobilization. Surfacetreatments using various kinds of silane coupling agents are alsoeffective. A probe in which a functional group which can react with afunctional group introduced by a silane coupling agent is used. A methodof applying a resin having a functional group can also be used.

The detection of pathogenic fungus DNA using the probe carrier of thepresent invention can be carried out by a DNA-detecting method at leastincluding:

(i) reacting a sample with the probe carrier on which the probe of thepresent invention is immobilized; and

(ii) detecting the reaction intensity of the probe on the probe carrierwhich is reacted with the nucleic acid contained in the sample.

In addition, the detection can be carried out by a detecting method atleast including:

(a) reacting a sample with the probe carrier on which the probe of thepresent invention is immobilized;

(b) detecting the reaction intensity of the probe on the probe carrierwith a nucleic acid in the sample; and

(c) specifying the probe reacted with the nucleic acid in the samplewhen the reaction of the probe with the nucleic acid in the sample isdetected and specifying the DNA of a pathogenic fungus in the samplebased on the base sequence of the probe.

The probe to be immobilized on the probe carrier is at least one of theabove-mentioned items (A) to (P). On the carrier, other probes (thosefor detecting fungus species other than Candida kefyr) may beimmobilized depending on the purpose of test. In this case, the otherprobes may be those capable of detecting the fungus species other thanCandida kefyr without causing cross contamination and the use of suchprobes allows simultaneous detection of a plurality of fungus specieswith high accuracy.

As described above, when the base sequence of the ITS region on the DNAsequence of the pathogenic fungus contained in the specimen beingamplified by PCR is used as a sample to be reacted with the probecarrier, the primer set for detecting the pathogenic fungus can be used.The primer set is preferably one containing oligonucleotides having thefollowing known base sequences:

(1) 5′ tccgtaggtgaacctgcgg 3′ (ITS1); (SEQ ID NO. 5) and (2)5′ tcctccgcttattgatatgc 3′ (ITS4). (SEQ ID NO. 6)

Accordingly, the detection method of the present invention may furtherinclude amplifying the target nucleic acid in the specimen by PCR usingoligonucleotides having the above-mentioned base sequences (1) and (2)as primers.

A kit for detecting a pathogenic fungus can be prepared using at leastany of the probes as described above and a reagent for detecting areaction of the probe with nucleic acid in a sample. The probe in such akit may be provided as the probe carrier. In addition, the reagent forthe detection may contain a primer to be used in labeling for detectingthe detection or used in amplification as a pretreatment. In the casewhere the detection reagent contains the primer, the primer ispreferably one suitable for amplifying the DNA of the ITS region ofCandida kefyr. Further, the detection reagent may contain a primer forapplying the DNA of the ITS region of any pathogenic fungus other thanCandida kefyr in addition to the primer for amplifying the DNA of theITS region of Candida kefyr.

EXAMPLES

Hereinafter, the present invention is described in further details withreference to examples using probes for detecting pathogenic fungi.

Example 1 Production of DNA Chip

In this example, the production of a DNA chip on which the probe of thepresent invention is immobilized is described.

(1. Preparation of Probe DNA)

First, nucleic acid sequences represented in Table 1 were designed asprobes for detecting fungi. Specifically, the probe base sequencesrepresented as follows were selected from the ITS regions on the fungalDNA sequences represented in Table 1. Those probe base sequences aredesigned so that they will be expected to have extremely highspecificities and sufficient hybridization sensitivities without anyvariation in the respective probe base sequences. Note that the probebase sequences do not have to be limited to the sequences whichcompletely correspond to those represented in Table 1. The basesequences having about 20 to 30 bases each of which include the probebase sequences may be also included in the probe base sequences ofTable 1. In this case, other portions other than the base sequences asdefined in Table 1 should have base sequences which do not affect thedetection accuracy.

TABLE 1 Probe sequence SEQ Name of Probe ID fungus name NO. Basesequence Candida P0601 1 5′ gcggccagttcttgattctctgc 3′ kefyr P0602 25′ agctcgtctctccagtggacataaac 3′ P0603 3 5′ ttgaaagtggctagccgttgcc 3′P0604 4 5′ tcgtggtaagcttgggtcatagagac 3′

In each of the probes shown in Table 1, a thiol group was introduced onthe 5′ terminal of the nucleic acid as a functional group to beimmobilized on a DNA chip in accordance with the conventionalprocedures. After the introduction of the functional group, the probewas purified and then freeze-dried. Subsequently, the freeze-dried probewas stored in a refrigerator at −30° C.

(2. Preparation of PCR Primer)

(2-1. Preparation of PCR Primer for Specimen Amplification)

Conventional primers are used as PCR primers for amplifying the ITSregion on DNA to be used for detecting a pathogenic fungus. Theconventional primers are shown in Table 2. Specifically, theconventional primers are provided as a set of conventional primers forspecifically amplifying the ITS region of DNA, designed from regionscommon to fungi so as to carry out simultaneous amplification even whenfungus species are different.

TABLE 2 Primer sequence SEQ Primer Primer ID type name NO. SequenceForward ITS1 5 5′ tccgtaggtgaacctgcgg 3′ Primer Reverse ITS4 65′ tcctccgcttattgatatgc 3′ Primer

The primers shown in Table 2 were synthesized and then purified byhigh-performance liquid chromatography (HPLC). Subsequently, each primerwas dissolved in a TE buffer to obtain the primer at a finalconcentration of 10 pmol/μl.

(2-2. Preparation of PCR Primer for Labeling)

A primer for labeling is prepared by introducing a label into a reverseprimer of the primer for specimen amplification. The primer for labelingis shown in Table 3.

TABLE 3 Labeled primer sequence SEQ Primer ID type Primer name NO.Sequence Labeled Cy3-labeled- 7 5′ tcctccgcttattgatatgc 3′ primer ITS4

The primer shown in Table 3 was labeled with fluorescent dye Cy3. Afterthe synthesis, the primer was purified by high-performance liquidchromatography (HPLC) and then dissolved in a TE buffer to obtain theprimer at a final concentration of 10 pmol/μl.

(3. Production of DNA Chip)

(3-1. Cleaning of Glass Substrate)

A glass substrate (size: 25 mm×75 mm×1 mm, manufactured by IIYAMAPRECISION GLASS CO., LTD.) formed of synthetic quartz was placed in aheat- and alkali-resisting rack and immersed in a cleaning solution forultrasonic cleaning, which was adjusted to have a predeterminedconcentration. The glass substrate was kept immersed in the cleaningsolution for a night and cleaned by ultrasonic cleaning for 20 minutes.The substrate was picked up, lightly rinsed with pure water, and cleanedby ultrasonic cleaning in ultrapure water for 20 minutes. The substratewas immersed in a 1N aqueous sodium hydroxide solution heated to 80° C.for 10 minutes. Pure water cleaning and ultrapure water cleaning wereexecuted again. A quartz glass substrate for a DNA chip was thusprepared.

(3-2. Surface Treatment)

A silane coupling agent KBM-603 (manufactured by Shin-Etsu Chemical Co.,LTd.) was dissolved in pure water at a concentration of 1% by weight (wt%) and stirred at room temperature for 2 hours. Subsequently, thecleaned glass substrate was immersed in the aqueous solution of thesilane coupling agent and left standing at room temperature for 20minutes. The glass substrate was picked up. The surface thereof waslightly rinsed with pure water and dried by spraying nitrogen gas toboth surfaces of the substrate. The dried substrate was baked in an ovenat 120° C. for 1 hour to complete the coupling agent treatment, wherebyan amino group was introduced to the substrate surface. Next,N-maleimidecaproyloxy succinimide (hereinafter abbreviated as EMCS) wasdissolved in a 1:1 (volume ratio) solvent mixture of dimethyl sulfoxideand ethanol to obtain a final concentration of 0.3 mg/ml to obtain anEMCS solution. As EMCS, N-(6-maleimidecaproyloxy)succinimidemanufactured by Dojindo Laboratories was used.

The baked glass substrate was left standing and cooled and immersed inthe prepared EMCS solution at room temperature for 2 hours. By thistreatment, the amino group introduced to the surface of the substrate bythe silane coupling agent reacted with the succinimide group in the EMCSto thereby introduce the maleimide group to the surface of the glasssubstrate. The glass substrate picked up from the EMCS solution wascleaned by using the above-mentioned solvent mixture in which the EMCSwas dissolved. The glass substrate was further cleaned by ethanol anddried in a nitrogen gas atmosphere.

(3-3. Probe DNA)

The microorganism detection probe prepared in the item (1. Preparationof probe DNA) of Example 1 was dissolved in pure water. The solution wasdispensed such that the final concentration (at ink dissolution) was 10μM. Then, the solution was freeze-dried to remove water.

(3-4. DNA Discharge by BJ Printer and Bonding to Substrate)

An aqueous solution containing 7.5-wt % glycerin, 7.5-wt % thiodiglycol,7.5-wt % urea, and 1.0-wt % Acetylenol EH (manufactured by Kawaken FineChemicals Co., Ltd.) was prepared. Each of the 4 probes (Table 1)prepared in advance was dissolved in the solvent mixture at apredetermined concentration. An ink tank for an inkjet printer (tradename: BJF-850, manufactured by Canon Inc.) is filled with the resultantDNA solution and attached to the printhead. Note that the inkjet printerused here was modified in advance to allow printing on a flat plate.When a printing pattern is input in accordance with a predetermined filecreation method, about 5-picoliter of a DNA solution can be spotted at apitch of about 120 μm. Subsequently, the printing operation was executedfor one glass substrate by using the modified inkjet printer to preparean array. After confirming that printing was reliably executed, theglass substrate was left standing in a humidified chamber for 30 minutesto make the maleimide group on the glass substrate surface react withthe thiol group at the nucleic acid probe terminal.

(3-5. Cleaning)

After reaction for 30 minutes, the DNA solution remaining on the surfacewas cleaned by using a 10-mM phosphate buffer (pH 7.0) containing 100-mMNaCl, thereby obtaining a DNA chip in which single-stranded DNAs wereimmobilized on the glass substrate surface.

Example 2 Detection of Candida kefyr

In this example, the detection of a microorganism using a two-step PCRmethod is described.

(1. Extraction of DNA from Candida kefyr (Model Specimen))

(1-1. Culture of Microorganism and DNA Extraction Therefrom)

First, Candida kefyr (standard strain, ATCC 42265) was cultured inaccordance with a conventional method. Subsequently, DNA was extractedfrom the microorganism culture medium and then purified by a nucleicacid purification kit (FastPrep FP100A and Fast DNA Kit, manufactured byFunakoshi Co., Ltd.).

(1-2. Examination of Collected DNA)

The collected DNA of the microorganism (Candida kefyr) was subjected toagarose electrophoresis and absorbance determination at 260/280 nm toexamine the qualities of the DNA (i.e., the amount of low-molecularweight nucleic acid as a contaminant and the degree of decomposition)and the collected amount of the DNA. In this example, about 10 μg of DNAwas collected, while the degradation of DNA and the contamination ofribosomal RNA were not recognized. The collected DNA was dissolved in aTE buffer to a final concentration of 50 ng/μl and then used in theexample described below.

(2. Amplification and Labeling of Specimen)

(2-1. Amplification of Specimen: 1st PCR)

An amplification (1st PCR) reaction of microorganism DNA as an analyteis shown in Table 1 below. The amplification reaction employed theprimer set shown in the item (2-1. Culture of microorganism and DNAextraction therefrom) of Example 1, as described above.

TABLE 4 Composition of 1st PCR reaction solution TaKaRa ExTaq 25.0 μLPrimer mix 2.0 μL Forward primer 1.0 μL Reverse primer 1.0 μL Template1.0 μL Water up to 50 μL Total 50 μL

The amplification reaction was carried out by a commercially-availablethermal cycler with the reaction solution of the above-mentionedcomposition in accordance with the protocol illustrated in FIG. 1.

After completing the reaction, the amplified product was purified usinga purification column (QIAquick PCR Purification Kit manufactured byQIAGEN), followed by the quantitative assay of the amplified product.

(2-2. Labeling Reaction: 2nd PCR)

A reaction solution of the composition shown in Table 5 was subjected toan amplification reaction with a commercially-available thermal cyclerin accordance with the protocol illustrated in FIG. 2. The amplificationreaction employed the primer set shown in the item (2-2. Preparation ofPCR primer for labeling) of Example 1, as described above.

TABLE 5 Composition of PCR reaction solution TaKaRa ExTaq 25.0 μLLabeled primer 5.0 μL Template DNA variable (1st PCR Product) (30ng/tube) Water up to 50 μL Total 50 μL

After completing the reaction, the primer was purified using apurification column (QIAquick PCR Purification Kit manufactured byQIAGEN) to obtain a labeled specimen.

(3. Hybridization)

Detection reaction was performed using the DNA chip prepared in the item(3. Preparation of DNA chip) of Example 1 and the labeled specimenprepared in the item (2. Amplification and labeling of specimen) ofExample 2.

(3-1. Blocking of DNA Chip)

Bovine serum albumin (BSA, Fraction V: manufactured by Sigma) wasdissolved in a 100-mM NaCl/10-mM phosphate buffer such that a 1 wt %solution was obtained. Then, the DNA chip prepared in the item (3.Preparation of DNA chip) of Example 1 was immersed in the solution atroom temperature for 2 hours to execute blocking. After completing theblocking, the chip was cleaned using a washing solution as describedbelow, rinsed with pure water and hydro-extracted by a spin dryer.

Washing Solution:

2×SSC solution (300 mM of NaCl, 30 mM of sodium citrate (trisodiumcitrate dihydrate, C6H₅Na₃.2H₂O), pH 7.0) containing 0.1-wt % sodiumdodecyl sulfate (SDS).

(3-2. Hybridization)

A drained DNA chip was set on a hybridization device (HybridizationStation, manufactured by Genomic Solutions Inc.) and a hybridizationreaction was then carried out using a hybridization solution shown inTable 6 below in accordance with the protocol illustrated in FIG. 3.

TABLE 6 Composition of hybridization solution 20 X SSPE 39.0 μLformamide 13.0 μL 25 nM positive control  1.3 μL water 13.7 μL TemplateDNA (2nd PCR product) 50.0 μL 0.5% SDS 13.0 μL Total 130.0 μL 

(4. Detection of Microorganism (Fluorescent Assay))

The DNA chip after the above-mentioned hybridization reaction wassubjected to a fluorescent assay using a DNA-chip fluorescence detector(GenePix 400B, manufactured by Axon Co., Ltd.). Consequently, Candidakefyr could be detected with good reproducibility and sufficient signalintensity. The measurement results of Candida kefyr are shown in Table 7below.

TABLE 7 Measurement results of fluorescence intensity: Candida kefyrProbe SEQ ID 1st time 2nd time name NO. Intensity S/N Intensity S/NP0601 1 7074.0 87.3 2535.2 55.8 P0602 2 12223.7 150.9 4219.5 92.9 P06033 9000.8 111.1 2951.8 65.0 P0604 4 31074.6 383.7 14120.9 310.8 BG — 81.0— 45.4 — average

As shown in Table 7 above, the first to fourth probes for Candida kefyrshown in Table 1 expressed specific hybridization and it was confirmedthat there was a nucleic acid sequence in the DNA extract from themicroorganism culture medium which has the same sequence as that of theITS region of the DNA of Candida kefyr. Therefore, it can be concludedthat the DNA chip will allow the detection of Candida kefyr.

(5. Results)

As is evident from the above-mentioned description, the above-mentionedexamples allowed the preparation of a DNA chip on which a probe set,which was able to detect Candida kefyr, was immobilized. Further, theuse of the DNA chip allowed the identification of a pathogenic fungus,so the problems of the DNA probe derived from a microorganism wassolved. In other words, the oligonucleotide probe can be chemicallyproduced in large amounts, while the purification or concentrationthereof can be controlled. For the purpose of classifying the species ofmicroorganisms, a probe set capable of collectively detecting the samefungus species could be provided. In addition, according to theembodiment, the base sequence of the ITS region on the DNA sequence ofthe pathogenic fungus can be sufficiently detected and thus the presenceof the pathogenic fungus can be effectively determined with highaccuracy.

Example 3 Experiment of Other Fungi

This example describes that strong hybridization cannot be detected onany of fungi other than Candida kefyr when the DNA chip prepared inExample 1 is employed.

(1. Extraction of DNA (Model Specimen))

(1-1. Culture of Microorganism and DNA Extraction Therefrom)

The following fungal strains were cultured in a manner similar toExample 2 and the DNA thereof was then extracted and purified. Thedeposit number of each fungus strain is represented in the parenthesesafter the name of the fungus.

Candida dubliniensis (ATCC MYA-646)

Candida glabrata (JCM 3761)

Candida guilliermondii (ATCC 6260)

Candida intermedia (ATCC 14439)

Candida kefyr (ATCC 42265)

Candida krusei (JCM 1609)

Candida lusitaniae (ATCC 34449)

Candida parapsilosis (JCM 1618)

Candida tropicalis (JCM 1541)

Trichosporon cutaneum (JCM 1462)

Trichosporon asahii (JCM 1809)

Cryptococcus neoformans (ATCC 32045)

Aspergillus fumigatus (JCM 10253)

Aspergillus niger (JCM 10254)

Epidermophyton floccosum (ATCC 52063)

Arthroderma otae (ATCC 28327)

Arthroderma gypseum (ATCC 24163)

Arthroderma benhamiae (ATCC 16781)

Trichophyton rubrum (ATCC 10218)

Trichophyton tonsurans (ATCC 10217)

Trichophyton verrucosum (ATCC 28203)

Trichophyton violaceum (ATCC 28944)

Arthroderma vanbreuseghemii (ATCC 28145)

Arthroderma incurvatum (ATCC 24005)

Trichophyton interdigitale (IFM 55365)

(1-2. Examination of Collected DNA)

The collected DNAs of the respective fungi were subjected to the assayof the collected amount thereof as described in Example 2. The collectedDNA was dissolved in a TE buffer to have a final concentration of 50ng/μl and then used in the following example.

(2. Amplification and Labeling of Specimen)

(2-1. Amplification of Specimen: 1st PCR)

An amplification reaction of microorganism DNA to be provided as aspecimen was carried out in a manner similar to that of the item (2-1.Amplification of specimen: 1st PCR) of Example 2, as described above.After completing the reaction, the amplified product was purified usinga purification column (QIAquick PCR Purification Kit manufactured byQIAGEN), followed by the quantitative assay of the amplified product.

(2-2. Labeling Reaction: 2nd PCR)

A labeling reaction was carried out using the amplified product obtainedin the item (2-1. Amplification of specimen: 1st PCR) in a mannersimilar to that of the item (2-2. Labeling reaction: 2nd PCR) of Example2, as described above. After completing the reaction, the labeledproduct was purified using a purification column (QIAquick PCRPurification Kit manufactured by QIAGEN), thereby obtaining a labeledspecimen.

(3. Hybridization)

A detection reaction was carried out using the DNA chip manufactured inthe item (3. Manufacture of DNA chip) of Example 1, as described aboveand the labeled specimen prepared in the item (2. Amplification andlabeling of specimen) in a manner similar to the item (3. Hybridization)of Example 2, as described above.

(4. Detection of Microorganism (Fluorescent Assay))

A fluorescent assay was carried out in a manner similar to the item (4.Detection of microorganism (fluorescent assay)) of Example 2 asdescribed above. The measurement results of the respective fungalspecies are shown in Tables 8 to 32 below.

TABLE 8 Measurement results of fluorescence intensity: Candida albicansProbe SEQ ID 1st time 2nd time name NO. Intensity S/N Intensity S/NP0601 1 80.8 0.9 44.0 0.9 P0602 2 73.7 0.9 43.2 0.9 P0603 3 76.5 0.944.4 0.9 P0604 4 79.8 0.9 46.0 0.9 BG — 86.0 — 49.0 — average

As shown in Table 8 above, in the probe set for Candida kefyr shown inTable 1, it is evident that any specific hybridization does not occur inall probes, compared with the experimental results of Table 7 above. Itcan be concluded that the DNA chip scarcely cross-hybridize to Candidaalbicans.

TABLE 9 Measurement results of fluorescence intensity: Candidadubliniensis Probe SEQ ID 1st time 2nd time name NO. Intensity S/NIntensity S/N P0601 1 74.8 0.7 44.3 0.9 P0602 2 75.3 0.7 43.6 0.9 P06033 69.2 0.6 42.7 0.9 P0604 4 68.8 0.6 44.3 0.9 BG — 109.9 — 46.7 —average

As shown in Table 9 above, in the probe set for Candida kefyr shown inTable 1, it is evident that any specific hybridization does not occur inall probes, compared with the experimental results of Table 7 above. Itcan be concluded that the DNA chip scarcely cross-hybridize to Candidadubliniensis.

TABLE 10 Measurement results of fluorescence intensity: Candida glabrataProbe SEQ ID 1st time 2nd time name NO. Intensity S/N Intensity S/NP0601 1 88.6 0.9 42.6 1.0 P0602 2 94.0 0.9 42.6 1.0 P0603 3 86.8 0.841.3 1.0 P0604 4 88.8 0.9 42.7 1.0 BG — 102.8 — 43.4 — average

As shown in Table 10 above, in the probe set for Candida kefyr shown inTable 1, it is evident that any specific hybridization does not occur inall probes, compared with the experimental results of Table 7 above. Itcan be concluded that the DNA chip scarcely cross-hybridize to Candidaglabrata.

TABLE 11 Measurement results of fluorescence intensity: Candidaguilliermondii Probe SEQ ID 1st time 2nd time name NO. Intensity S/NIntensity S/N P0601 1 65.2 0.7 47.2 1.0 P0602 2 65.6 0.7 45.1 0.9 P06033 63.3 0.7 45.5 0.9 P0604 4 63.5 0.7 48.7 1.0 BG — 92.8 — 48.4 — average

As shown in Table 11 above, in the probe set for Candida kefyr shown inTable 1, it is evident that any specific hybridization does not occur inall probes, compared with the experimental results of Table 7 above. Itcan be concluded that the DNA chip scarcely cross-hybridize to Candidaguilliermondii.

TABLE 12 Measurement results of fluorescence intensity: Candidaintermedia Probe SEQ ID 1st time 2nd time name NO. Intensity S/NIntensity S/N P0601 1 65.6 0.8 47.5 1.0 P0602 2 58.4 0.7 44.3 0.9 P06033 60.3 0.7 44.8 0.9 P0604 4 57.4 0.7 44.9 0.9 BG — 81.4 — 48.2 — average

As shown in Table 12 above, in the probe set for Candida kefyr shown inTable 1, it is evident that any specific hybridization does not occur inall probes, compared with the experimental results of Table 7 above. Itcan be concluded that the DNA chip scarcely cross-hybridize to Candidaintermedia.

TABLE 13 Measurement results of fluorescence intensity: Candida kruseiProbe SEQ ID 1st time 2nd time name NO. Intensity S/N Intensity S/NP0601 1 109.1 0.6 44.7 0.9 P0602 2 102.9 0.6 44.3 0.9 P0603 3 113.3 0.745.9 0.9 P0604 4 108.8 0.6 51.0 1.0 BG — 168.8 — 49.8 — average

As shown in Table 13 above, in the probe set for Candida kefyr shown inTable 1, it is evident that any specific hybridization does not occur inall probes, compared with the experimental results of Table 7 above. Itcan be concluded that the DNA chip scarcely cross-hybridize to Candidakrusei.

TABLE 14 Measurement results of fluorescence intensity: Candidalusitaniae Probe SEQ ID 1st time 2nd time name NO. Intensity S/NIntensity S/N P0601 1 76.3 0.8 47.9 0.9 P0602 2 83.5 0.9 46.3 0.9 P06033 78.9 0.9 46.7 0.9 P0604 4 86.3 0.9 47.5 0.9 BG — 92.3 — 52.1 — average

As shown in Table 14 above, in the probe set for Candida kefyr shown inTable 1, it is evident that any specific hybridization does not occur inall probes, compared with the experimental results of Table 7 above. Itcan be concluded that the DNA chip scarcely cross-hybridize to Candidalusitaniae.

TABLE 15 Measurement results of fluorescence intensity: Candidaparapsilosis Probe SEQ ID 1st time 2nd time name NO. Intensity S/NIntensity S/N P0601 1 65.7 0.8 42.0 0.9 P0602 2 64.9 0.8 43.0 0.9 P06033 66.3 0.8 42.7 0.9 P0604 4 60.1 0.7 45.8 0.9 BG — 83.1 — 48.4 — average

As shown in Table 15 above, in the probe set for Candida kefyr shown inTable 1, it is evident that any specific hybridization does not occur inall probes, compared with the experimental results of Table 7 above. Itcan be concluded that the DNA chip scarcely cross-hybridize to Candidaparapsilosis.

TABLE 16 Measurement results of fluorescence intensity: Candidatropicalis Probe SEQ ID 1st time 2nd time name NO. Intensity S/NIntensity S/N P0601 1 58.9 0.7 42.4 0.9 P0602 2 60.5 0.7 41.9 0.9 P06033 62.9 0.8 42.0 0.9 P0604 4 59.0 0.7 44.5 0.9 BG — 81.8 — 47.6 — average

As shown in Table 16 above, in the probe set for Candida kefyr shown inTable 1, it is evident that any specific hybridization does not occur inall probes, compared with the experimental results of Table 7 above. Itcan be concluded that the DNA chip scarcely cross-hybridize to Candidatropicalis.

TABLE 17 Measurement results of fluorescence intensity: Trichosporoncutaneum Probe SEQ ID 1st time 2nd time name NO. Intensity S/N IntensityS/N P0601 1 73.1 0.8 49.1 1.0 P0602 2 66.8 0.8 45.8 0.9 P0603 3 68.6 0.845.2 0.9 P0604 4 67.0 0.8 51.1 1.0 BG — 87.9 — 50.8 — average

As shown in Table 17 above, in the probe set for Candida kefyr shown inTable 1, it is evident that any specific hybridization does not occur inall probes, compared with the experimental results of Table 7 above. Itcan be concluded that the DNA chip scarcely cross-hybridize toTrichosporon cutaneum.

TABLE 18 Measurement results of fluorescence intensity: Trichosporonasahii Probe SEQ ID 1st time 2nd time name NO. Intensity S/N IntensityS/N P0601 1 73.8 0.8 48.8 0.9 P0602 2 84.3 0.9 46.5 0.9 P0603 3 77.1 0.946.9 0.9 P0604 4 67.4 0.7 47.9 0.9 BG — 90.0 — 53.8 — average

As shown in Table 18 above, in the probe set for Candida kefyr shown inTable 1, it is evident that any specific hybridization does not occur inall probes, compared with the experimental results of Table 7 above.Consequently, It can be concluded that the DNA chip scarcelycross-hybridize to Trichosporon asahii.

TABLE 19 Measurement results of fluorescence intensity: Cyptococcusneoformans Probe SEQ ID 1st time 2nd time name NO. Intensity S/NIntensity S/N P0601 1 92.8 0.8 43.6 1.0 P0602 2 93.0 0.8 44.3 1.0 P06033 94.3 0.8 44.7 1.0 P0604 4 91.7 0.8 56.0 1.2 BG — 117.2 — 45.2 —average

As shown in Table 19 above, in the probe set for Candida kefyr shown inTable 1, it is evident that any specific hybridization does not occur inall probes, compared with the experimental results of Table 7 above. Itcan be concluded that the DNA chip scarcely cross-hybridize toCyptococcus neoformans.

TABLE 20 Measurement results of fluorescence intensity: Aspergillusfumigatus Probe SEQ ID 1st time 2nd time name NO. Intensity S/NIntensity S/N P0601 1 76.4 0.7 55.1 0.8 P0602 2 81.8 0.8 55.5 0.8 P06033 75.2 0.7 55.4 0.8 P0604 4 76.4 0.7 54.7 0.7 BG — 106.8 — 73.5 —average

As shown in Table 20 above, in the probe set for Candida kefyr shown inTable 1, it is evident that any specific hybridization does not occur inall probes, compared with the experimental results of Table 7 above. Itcan be concluded that the DNA chip scarcely cross-hybridize toAspergillus fumigatus.

TABLE 21 Measurement results of fluorescence intensity: Aspergillusniger Probe SEQ ID 1st time 2nd time name NO. Intensity S/N IntensityS/N P0601 1 89.4 0.8 48.8 1.0 P0602 2 102.3 0.9 46.2 0.9 P0603 3 83.90.7 46.0 0.9 P0604 4 91.7 0.8 51.0 1.0 BG — 119.1 — 50.7 — average

As shown in Table 21 above, in the probe set for Candida kefyr shown inTable 1, it is evident that any specific hybridization does not occur inall probes, compared with the experimental results of Table 7 above. Itcan be concluded that the DNA chip scarcely cross-hybridize toAspergillus niger.

TABLE 22 Measurement results of fluorescence intensity: Epidermophytonfloccosum Probe SEQ ID 1st time 2nd time name NO. Intensity S/NIntensity S/N P0601 1 86.4 1.0 47.5 0.9 P0602 2 85.1 0.9 46.8 0.9 P06033 83.5 0.9 48.3 0.9 P0604 4 81.5 0.9 47.4 0.9 BG — 89.7 — 53.0 — average

As shown in Table 22 above, in the probe set for Candida kefyr shown inTable 1, it is evident that any specific hybridization does not occur inall probes, compared with the experimental results of Table 7 above. Itcan be concluded that the DNA chip scarcely cross-hybridize toEpidermophyton floccosum.

TABLE 23 Measurement results of fluorescence intensity: Arthroderma otaeProbe SEQ ID 1st time 2nd time name NO. Intensity S/N Intensity S/NP0601 1 78.3 0.8 46.1 0.9 P0602 2 75.6 0.8 45.3 0.9 P0603 3 76.1 0.846.8 0.9 P0604 4 75.2 0.8 48.9 1.0 BG — 94.2 — 49.5 — average

As shown in Table 23 above, in the probe set for Candida kefyr shown inTable 1, it is evident that any specific hybridization does not occur inall probes, compared with the experimental results of Table 7 above. Itcan be concluded that the DNA chip scarcely cross-hybridize toArthroderma otae.

TABLE 24 Measurement results of fluorescence intensity: Arthrodermagypseum Probe SEQ ID 1st time 2nd time name NO. Intensity S/N IntensityS/N P0601 1 76.7 0.8 48.4 0.9 P0602 2 79.4 0.9 47.7 0.9 P0603 3 79.5 0.949.2 0.9 P0604 4 78.3 0.9 49.6 0.9 BG — 90.4 — 53.0 — average

As shown in Table 24 above, in the probe set for Candida kefyr shown inTable 1, it is evident that any specific hybridization does not occur inall probes, compared with the experimental results of Table 7 above. Itcan be concluded that the DNA chip scarcely cross-hybridize toArthroderma gypseum.

TABLE 25 Measurement results of fluorescence intensity: Arthrodermabenhamiae Probe SEQ ID 1st time 2nd time name NO. Intensity S/NIntensity S/N P0601 1 87.3 0.8 58.0 0.9 P0602 2 86.8 0.8 57.5 0.9 P06033 86.8 0.8 56.1 0.9 P0604 4 95.3 0.9 87.3 1.3 BG — 107.7 — 64.9 —average

As shown in Table 25 above, in the probe set for Candida kefyr shown inTable 1, it is evident that any specific hybridization does not occur inall probes, compared with the experimental results of Table 7 above. Itcan be concluded that the DNA chip scarcely cross-hybridize toArthroderma benhamiae.

TABLE 26 Measurement results of fluorescence intensity: Trichophytonrubrum Probe SEQ ID 1st time 2nd time name NO. Intensity S/N IntensityS/N P0601 1 81.6 0.9 44.9 0.9 P0602 2 76.9 0.8 47.2 1.0 P0603 3 79.4 0.946.3 0.9 P0604 4 77.2 0.8 46.3 0.9 BG — 93.2 — 49.5 — average

As shown in Table 26 above, in the probe set for Candida kefyr shown inTable 1, it is evident that any specific hybridization does not occur inall probes, compared with the experimental results of Table 7 above. Itcan be concluded that the DNA chip scarcely cross-hybridize toTrichophyton rubrum.

TABLE 27 Measurement results of fluorescence intensity: Trichophytontonsurans Probe SEQ ID 1st time 2nd time name NO. Intensity S/NIntensity S/N P0601 1 84.7 0.9 44.8 0.9 P0602 2 77.8 0.8 45.4 0.9 P06033 88.3 0.9 44.8 0.9 P0604 4 83.9 0.8 46.0 0.9 BG — 99.1 — 48.4 — average

As shown in Table 27 above, in the probe set for Candida kefyr shown inTable 1, it is evident that any specific hybridization does not occur inall probes, compared with the experimental results of Table 7 above. Itcan be concluded that the DNA chip scarcely cross-hybridize toTrichophyton tonsurans.

TABLE 28 Measurement results of fluorescence intensity: Trichophytonverrucosum Probe SEQ ID 1st time 2nd time name NO. Intensity S/NIntensity S/N P0601 1 91.4 0.8 56.0 1.0 P0602 2 89.1 0.8 48.0 0.9 P06033 94.2 0.8 48.7 0.9 P0604 4 82.3 0.7 238.7 4.3 BG — 111.9 — 55.1 —average

As shown in Table 28 above, in the probe set for Candida kefyr shown inTable 1, it is evident that any specific hybridization does not occur inall probes, compared with the experimental results of Table 7 above. Itcan be concluded that the DNA chip scarcely cross-hybridize toTrichophyton verrucosum.

TABLE 29 Measurement results of fluorescence intensity: Trichophytonviolaceum Probe SEQ ID 1st time 2nd time name NO. Intensity S/NIntensity S/N P0601 1 90.9 0.8 49.9 0.9 P0602 2 104.8 0.9 49.0 0.9 P06033 81.2 0.7 48.0 0.9 P0604 4 92.0 0.8 70.0 1.3 BG — 116.6 — 52.5 —average

As shown in Table 29 above, in the probe set for Candida kefyr shown inTable 1, it is evident that any specific hybridization does not occur inall probes, compared with the experimental results of Table 7 above. Itcan be concluded that the DNA chip scarcely cross-hybridize toTrichophyton violaceum.

TABLE 30 Measurement results of fluorescence intensity: Arthrodermavanbreuseghemii Probe SEQ ID 1st time 2nd time name NO. Intensity S/NIntensity S/N P0601 1 72.5 0.7 51.7 1.0 P0602 2 79.6 0.8 48.4 0.9 P06033 74.7 0.8 49.8 1.0 P0604 4 81.3 0.8 149.6 2.9 BG — 99.4 — 52.4 —average

As shown in Table 30 above, in the probe set for Candida kefyr shown inTable 1, it is evident that any specific hybridization does not occur inall probes, compared with the experimental results of Table 7 above. Itcan be concluded that the DNA chip scarcely cross-hybridize toArthroderma vanbreuseghemii.

TABLE 31 Measurement results of fluorescence intensity: Arthrodermaincurvatum Probe SEQ ID 1st time 2nd time name NO. Intensity S/NIntensity S/N P0601 1 92.8 0.8 47.3 1.0 P0602 2 95.7 0.9 45.4 0.9 P06033 98.4 0.9 47.4 1.0 P0604 4 96.5 0.9 49.8 1.0 BG — 110.7 — 49.6 —average

As shown in Table 31 above, in the probe set for Candida kefyr shown inTable 1, it is evident that any specific hybridization does not occur inall probes, compared with the experimental results of Table 7 above. Itcan be concluded that the DNA chip leads low crow hybridization toArthroderma incurvatum.

TABLE 32 Measurement results of fluorescence intensity: Trichophytoninterdigitale Probe SEQ ID 1st time 2nd time name NO. Intensity S/NIntensity S/N P0601 1 153.3 1.4 98.3 0.8 P0602 2 133.9 1.0 86.9 0.7P0603 3 199.3 1.8 124.8 1.1 P0604 4 117.9 1.0 86.0 0.7 BG — 113.5 —117.2 — average

As shown in Table 32 above, in the probe set for Candida kefyr shown inTable 1, it is evident that any specific hybridization does not occur inall probes, compared with the experimental results of Table 7 above. Itcan be concluded that the DNA chip scarcely cross-hybridize toTrichophyton interdigitale.

As descried above, according to Tables 8 to 32, the DNA chip having alow possibility of accidentally detecting any fungus other than Candidakefyr, on which the probe set for Candida kefyr was immobilized, couldbe prepared. The use of the DNA chip allows the detection of Candidakefyr. Besides, a probe set which can detect Candida kefyr whiledistinguishing it from other fungus species could be provided. Further,it has been described that the DNA chip produced in Example 1 can onlyspecifically hybridize with Candida kefyr but not hybridize with any ofother fungus species. Therefore, in a DNA chip which is prepared as acombination of the probe for Candida kefyr as described in Example 1 anda probe for other fungus species designed by the same idea as that ofthe former, a probe set can be provided so that it can not only detectCandida kefyr but also selectively detect Candida kefyr even in aspecimen containing a mixture of Candida kefyr with other fungusspecies.

The present invention is not limited to the above embodiments andvarious changes and modifications can be made within the spirit andscope of the present invention. Therefore to apprise the public of thescope of the present invention, the following claims are made.

While the present invention has been described with reference toexemplary embodiments, it is to be understood that the invention is notlimited to the disclosed exemplary embodiments. The scope of thefollowing claims is to be accorded the broadest interpretation so as toencompass all such modifications and equivalent structures andfunctions.

This application claims the benefit of Japanese Patent Application No.2007-128627, filed May 14, 2007, which is hereby incorporated byreference herein in its entirety.

1. A probe set for detecting a DNA of Candida kefyr which is apathogenic fungus, comprising four probes consisting of the followingbase sequences (1) to (4) respectively to specifically detect Candidakefyr: (1) gcggccagttcttgattctctgc (SEQ ID NO. 1) or the complementarysequence thereof; (2) agctcgtctctccagtggacataaac (SEQ ID NO. 2) or thecomplementary sequence thereof; (3) ttgaaagtggctagccgttgcc (SEQ ID NO.3) or the complementary sequence thereof; and (4)tcgtggtaagcttgggtcatagagac (SEQ ID NO. 4) or the complementary sequencethereof.
 2. A probe set for detecting a DNA of Candida kefyr which is apathogenic fungus, comprising the following probes (A) to (D), whereinthe probe set does not comprise another probe to detect Candida kefyr:(A) a probe consisting of gcggccagttcttgattctctgc (SEQ ID NO. 1); (B) aprobe consisting of agctcgtctctccagtggacataaac (SEQ ID NO. 2); (C) aprobe consisting of ttgaaagtggctagccgttgcc (SEQ ID NO. 3); and (D) aprobe consisting of tcgtggtaagcttgggtcatagagac (SEQ ID NO. 4).
 3. A kitfor specifically detecting a DNA of Candida kefyr, comprising: the probeset according to claim 1; and a reagent for detecting a reaction of aprobe with a target nucleic acid.
 4. A kit for specifically detecting aDNA of Candida kefyr, comprising: the probe set according to claim 2;and a reagent for detecting a reaction of a probe with a target nucleicacid.